We thank Dr. Yves L. Janin for his fruitful comments and suggestions. We would like to thank H.H. Gad for CHIK/Ren and C. Combredet for her technical support. This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Sant&#233; Et de la Recherche M&#233;dicale (INSERM), the Institut Carnot - Pasteur Maladies Infectieuses (Programme STING to P.O.V and H.M.-L), the Agence Nationale pour la Recherche (ANR-RPIB, Programme STING 2.0 to P.O.V), and the &#34;Conseil R&#233;gional d'Ile-de-France&#34; (Chemical Library Project, grants n&deg; I 06-222/R and I 09-1739/R to H.M.-L.). The work on CHIKV/Ren was supported by the project ArbOAS (ANR grant 2010-INTB-1601-02).

1. Preparation of 96-well Daughter Plates with Compounds
<ol>
	<li>Move 96-well mother plates containing 10 mM stock solutions of chemical compounds in DMSO from -20 &#176;C to room temperature. Dilute compounds 5x&#160;in DMSO to obtain intermediate 96-well dilution plates at 2 mM. Dilution is achieved by pipetting 5 &#956;l&#160;into 20 &#956;l of DMSO and mixing.</li>
	<li>Pipette 1 &#956;l from dilution plates into dry wells of white, bar-coded tissue culture 96-well plates. This first set of daughter plates (D1) w...

<ol>
	<li>De Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiviral+drugs+in+current+clinical+use.">Antiviral drugs in current clinical use.</a> J Clin Virol. 30, 115-133 (2004).</li><li>Debing, Y., Jochmans, D., Neyts, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervention+strategies+for+emerging+viruses:+use+of+antivirals.">Intervention strategies for emerging viruses: use of antivirals.</a> Curr Opin Virol. , (2013).</li><li>Leyssen, P., De Clercq, E., Neyts, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+strategies+to+inhibit+the+replication+of+RNA+viruses.">Molecular strategies to inhibit the replication of RNA viruses.</a> Antiviral Res. 78, 9-25 (2008).</li><li>Leyssen, P., Balzarini, J., De Clercq, E., Neyts, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+predominant+mechanism+by+which+ribavirin+exerts+its+antiviral+activity+in+vitro+against+flaviviruses+and+paramyxoviruses+is+mediated+by+inhibition+of+IMP+dehydrogenase.">The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase.</a> J Virol. 79, 1943-1947 (2005).</li><li>Wang, B. X., Fish, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yin+and+yang+of+viruses+and+interferons.">The yin and yang of viruses and interferons.</a> Trends Immunol. 33, 190-197 (2012).</li><li>Empey, K. M., Peebles, R. S., Kolls, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+advances+in+the+treatment+and+prevention+of+respiratory+syncytial+virus.">Pharmacologic advances in the treatment and prevention of respiratory syncytial virus.</a> Clin Infect Dis. 50, 1258-1267 (2010).</li><li>Tan, S. L., Ganji, G., Paeper, B., Proll, S., Katze, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+Toll-like+receptor+targeted+therapeutics.">New developments in Toll-like receptor targeted therapeutics.</a> Curr Opin Pharmacol. , (2012).</li><li>Thakur, C. 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The authors declare that they have no competing financial interests.

The screening pipeline described here aims at selecting compounds with a suitable profile as broad-spectrum inhibitors of RNA viruses. When a library of 10,000 compounds was screened with this protocol and aforementioned filtering criteria were applied, i.e. inhibition of MV replication superior to 75%, 40 compounds (0.4%) scored positive. Besides, about half of them showed some toxicity in the luciferase-based viability assay, and were disregarded for this reason. Finally, a dozen of hits readily available in l...

RNA viruses are responsible for a large variety of human infections, and have a huge impact on populations worldwide both in terms of public health and economical cost. Efficient vaccines have been developed against several human RNA viruses, and are widely used as prophylactic treatments. However, there is still a critical lack of therapeutic drugs against RNA virus infections. Indeed, efficient vaccines are not available against major human pathogens such as dengue virus, Hepatitis C virus or human respiratory syncytial virus (hRSV). Besides, RNA viruses are responsible for a majority of emerging diseases, which have increased in frequency because of global exchanges and human impact on ecological systems. Against this threat that represent RNA viruses, our therapeutic arsenal is extremely limited and relatively inefficient1-3. Current therapies are essentially based on recombinant type I interferons (IFN-&#945;/&#946;) to stimulate innate immunity, or the administration of ribavirin. Although the mode of action of this ribonucleoside analog is controversial and probably relies on various mechanisms, the inhibition of cellular IMPDH (inosine monophosphate dehydrogenase), which depletes intracellular GTP pools, is clearly essential4. Ribavirin, in combination with pegylated IFN-&#945;, is the main treatment against Hepatitis C virus. However, IFN-&#945;/&#946;&#160;and ribavirin treatments are of relatively poor efficacy in vivo against most RNA viruses as they efficiently blunt IFN-&#945;/&#946;&#160;signaling through expression of virulence factors5 and often escape ribavirin3. This added to the fact that ribavirin treatment is raising important toxicity issues, although it was recently approved against severe hRSV disease with controversial benefits6. More recently, some virus-specific treatments have been marketed, in particular against influenza virus with the development of neuraminidase inhibitors3. However, the large diversity and permanent emergence of RNA viruses precludes the development of specific treatments against each one of them in a relatively close future. Altogether, this stresses the need for efficient strategies to identify and develop potent antiviral molecules in the near future.
It is trivial to say that a broad-spectrum inhibitor active against a large panel of RNA viruses would solve this problem. Although such a molecule is still a virologist&#39;s dream, our better understanding of cellular defense mechanisms and innate immune system suggest that some possibilities exist7,8. Several academic and industrial laboratories are now seeking molecules that stimulate specific facets of cellular defense mechanisms or metabolic pathways to blunt viral replication. Although such compounds will probably show significant side effects, treatments against acute viral infections will be administered for a relatively short time, making them acceptable despite some potential toxicity on the long term. Various strategies have been developed to identify such broad-spectrum antiviral molecules. Some research programs aim at finding molecules that target specific defense or metabolic pathways. This includes, for example, pathogen recognition receptors to elicit antiviral gene expression9 and activate antiviral factors such as RNaseL10, autophagy machinery to promote virus degradation11, nucleoside synthesis pathways12,13, or apoptotic cascades to precipitate death of virus-infected cells14. Other groups have developed phenotypic screens that are not target-based13,15-17. In that case, antiviral molecules are simply identified by their capacity to block viral replication in a given cellular system. The general assumption is that a compound inhibiting 2-3 unrelated RNA viruses would have a suitable profile for a broad-spectrum antiviral molecule. The mode of action of hit compounds selected with such an empirical approach is only determined in a second time and eventually, may lead to the identification of novel cellular targets for antivirals. Interestingly, a retrospective analysis of new drugs approved by the US Food and Drug Administration between 1999 and 2008 has shown that in general, such phenotypic screenings tend to perform better than target-based approaches to discover first-in-class small-molecule drugs18.
Viral replication in high-throughput cell-based assays is usually determined from virus cytopathic effects. Cells are infected and cultured in 96- or 384-well plates in the presence of tested compounds. After few days, cellular layers are fixed and stained with dyes such as crystal violet. Finally, absorbance is determined with a plate reader and compounds inhibiting viral replication are identified by their capacity to preserve cellular layers from virus-induced cytopathic effect. Alternatively, virally-mediated cytopathic effects are assessed using standard viability assays such as MTS reduction. Such assays are highly tractable and cost-effective, but suffer from three major limitations. First, they require a virus-cell combination where viral replication is cytopathic in only few days but this is not always possible, thus calling for alternative approaches19. Second, they are poorly quantitative since they are based on an indirect measure of viral replication. Finally, toxic compounds can be scored as positive hits, and therefore must be eliminated with a counter screen measuring cellular viability. To overcome some of these hurdles, recombinant viruses or replicons have been engineered by reverse genetics to express reporter proteins, such as EGFP or luciferase, from an additional transcription unit or in frame with viral protein genes (few examples are 20-23). When these viruses replicate, reporter proteins are produced together with viral proteins themselves. This provides a very quantitative assay to measure viral replication and evaluate the inhibitory activity of candidate molecules. This is particularly true for recombinant viruses expressing luciferase (or other enzymes capable of bioluminescence) since this reporter system exhibits a wide dynamic range with a high sensitivity and virtually no background. Furthermore, there is no excitation light source, thus preventing interference with compound fluorescence24.
Here, we detail a high-throughput protocol to screen chemical libraries for broad-spectrum inhibitors of RNA viruses. Compounds are tested first on human cells infected with a recombinant measles virus (MV) expressing firefly luciferase25 (rMV2/Luc,&#160;Figure 1, primary screen). MV belongs to Mononegavirales order, and is often considered as a prototypical member of negative-strand RNA viruses. As such, MV genome is used as a template by the viral polymerase to synthesize mRNA molecules encoding for viral proteins. In the recombinant MV strain called rMV2/Luc, luciferase expression is expressed from an additional transcription unit inserted between P and M genes (Figure 2A). In parallel, compounds are tested for their toxicity on human cells using a commercial luciferase-based reagent that evaluates, by ATP quantification, the number of metabolically active cells in culture (Figure 1, primary screen). Entire chemical libraries can be easily screened with these two assays in order to select compounds that are not toxic and efficiently block MV replication. Then, hits are retested for dose-response inhibition of MV replication, the lack of toxicity as well as for their capacity to impair chikungunya virus (CHIKV) replication (Figure 1, secondary screen). CHIKV is a member of Togaviridae family and its genome is a positive single-strand RNA molecule. As such, it is completely unrelated to measles and compounds inhibiting both MV and CHIKV stand a great chance to inhibit a large panel of RNA viruses. CHIKV nonstructural proteins are directly translated from the viral genome, whereas structural proteins are encoded by transcription and translation of a subgenomic mRNA molecule. Our in vitro replication assay for CHIKV is based on a recombinant strain called CHIKV/Ren, which expresses Renilla luciferase&#160;enzyme as a cleaved part of the nonstructural polyprotein through an insertion of the reporter gene between nsP3 and nsP4 sequences26 (Figure 2B). The measure of Renilla luciferase activity allows the monitoring of viral replication at the early stage of CHIKV life cycle.
This high-throughput protocol was used to quickly identify compounds with a suitable profile for broad-spectrum antivirals in a commercial library of 10,000 molecules enriched for chemical diversity. Compounds were essentially following Lipinski&#39;s rule of five, with molecular weights ranging from 250 to 600 daltons, and log D values below 5. Most of these molecules were new chemical entities not available in other commercial libraries.

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Freedom EVO platform</td>
			<td style="width:105px;">TECAN</td>
			<td style="width:119px;"> </td>
			<td style="width:316px;">Robotic platform</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well Polystyrene Cell Culture Microplates, White</td>
			<td style="width:105px;">Greiner Bio One</td>
			<td style="width:119px;">655083</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellTiter-Glo Luminescent Cell Viability Assay</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">G7570</td>
			<td>Luciferase-based viability assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bright-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2610</td>
			<td>Reagent containing firefly luciferase substrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Renilla-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2710</td>
			<td>Reagent containing Renilla luciferase substrate</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Britelite plus Reporter Gene Assay System</td>
			<td style="width:105px;">Perkin-Elmer</td>
			<td style="width:119px;">6016761</td>
			<td style="width:316px;">Reagent containing firefly luciferase substrate. Can be used as an alternative to Brigh-Glo reagent to determine luciferase activity</td>
		</tr>
	</tbody>
</table>

high-throughput screening for broad-spectrum chemical inhibitors of rna viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

This screening pipeline relies first on the selection of compounds that inhibit MV replication and do not show any significant cellular toxicity (Figure 1, primary screen). It takes advantage of luciferase-based assays to determine cellular toxicity and the inhibition of viral replication. All pipetting steps and data acquisition can be performed in a high-throughput setting with a robotic platform.
Cellular toxicity of compounds is assessed using a luciferase-based viability ...

Watch this Scientific Journal Video about High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses at JoVE.com

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

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Research

JoVE Journal

Immunology and Infection

13.6K vistas.  Institut Pasteur, CNRS UMR3569.  En ensayos in vitro para medir la replicación del virus se han mejorado en gran medida por el desarrollo de virus de ARN recombinantes que expresan luciferasa u otras enzimas capaces de bioluminiscencia. A continuación te detallamos una tubería de cribado de alto rendimiento que combina dichas cepas recombinantes de virus del sarampión y chikungunya para aislar a los antivirales de amplio espectro de bibliotecas químicas. 